Mu Changyong, Zhong Qiwang, Meng Yan, Zhou Yong, Jiang Nan, Liu Wenzhi, Li Yiqun, Xue Mingyang, Zeng Lingbing, Vakharia Vikram N, Fan Yuding
Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Wuhan 430223, China.
College of Biological Science and Engineering, Jiangxi Agricultural University, Nanchang 330045, China.
Vaccines (Basel). 2021 Jan 12;9(1):41. doi: 10.3390/vaccines9010041.
The grass carp reovirus (GCRV) causes severe hemorrhagic disease with high mortality and leads to serious economic losses in the grass carp () industry in China. Oral vaccine has been proven to be an effective method to provide protection against fish viruses. In this study, a recombinant baculovirus BmNPV-VP35-VP4 was generated to express VP35 and VP4 proteins from GCRV type Ⅱ via Bac-to-Bac baculovirus expression system. The expression of recombinant VP35-VP4 protein (rVP35-VP4) in embryo cells (BmE) and silkworm pupae was confirmed by Western blotting and immunofluorescence assay (IFA) after infection with BmNPV-VP35-VP4. To vaccinate the grass carp by oral route, the silkworm pupae expressing the rVP35-VP4 proteins were converted into a powder after freeze-drying, added to artificial feed at 5% and fed to grass carp (18 ± 1.5 g) for six weeks, and the immune response and protective efficacy in grass carp after oral vaccination trial was thoroughly investigated. This included blood cell counting and classification, serum antibody titer detection, immune-related gene expression and the relative percent survival rate in immunized grass carp. The results of blood cell counts show that the number of white blood cells in the peripheral blood of immunized grass carp increased significantly from 14 to 28 days post-immunization (dpi). The differential leukocyte count of neutrophils and monocytes were significantly higher than those in the control group at 14 dpi. Additionally, the number of lymphocytes increased significantly and reached a peak at 28 dpi. The serum antibody levels were significantly increased at Day 14 and continued until 42 days post-vaccination. The mRNA expression levels of immune-related genes (IFN-1, TLR22, IL-1β, MHC I, Mx and IgM) were significantly upregulated in liver, spleen, kidney and hindgut after immunization. Four weeks post-immunization, fish were challenged with virulent GCRV by intraperitoneal injection. The results of this challenge study show that orally immunized group exhibited a survival rate of 60% and relative percent survival (RPS) of 56%, whereas the control group had a survival rate of 13% and RPS of 4%. Taken together, our results demonstrate that the silkworm pupae powder containing baculovirus-expressed VP35-VP4 proteins could induce both non-specific and specific immune responses and protect grass carp against GCRV infection, suggesting it could be used as an oral vaccine.
草鱼呼肠孤病毒（GCRV）可引发严重出血性疾病，死亡率高，给中国草鱼养殖业造成严重经济损失。口服疫苗已被证明是预防鱼类病毒的有效方法。在本研究中，通过杆状病毒表达系统（Bac-to-Bac）构建了重组杆状病毒BmNPV-VP35-VP4，以表达Ⅱ型GCRV的VP35和VP4蛋白。用BmNPV-VP35-VP4感染家蚕胚胎细胞（BmE）和蚕蛹后，通过蛋白质免疫印迹法（Western blotting）和免疫荧光分析（IFA）证实了重组VP35-VP4蛋白（rVP35-VP4）的表达。为通过口服途径给草鱼接种疫苗，将表达rVP35-VP4蛋白的蚕蛹冻干后制成粉末，按5%的比例添加到人工饲料中，投喂给体重为（18±1.5）g的草鱼，持续六周，并深入研究口服疫苗试验后草鱼的免疫反应和保护效果。这包括血细胞计数与分类、血清抗体效价检测、免疫相关基因表达以及免疫草鱼的相对存活率。血细胞计数结果显示，免疫草鱼外周血白细胞数量在免疫后14至28天显著增加。免疫后14天，中性粒细胞和单核细胞的分类白细胞计数显著高于对照组。此外，淋巴细胞数量显著增加，并在免疫后28天达到峰值。血清抗体水平在第14天显著升高，并持续至疫苗接种后42天。免疫后，肝脏、脾脏、肾脏和后肠中免疫相关基因（IFN-1、TLR22、IL-1β、MHC I、Mx和IgM）的mRNA表达水平显著上调。免疫四周后，通过腹腔注射用强毒GCRV对鱼进行攻毒。攻毒研究结果显示，口服免疫组的存活率为60%，相对存活率（RPS）为56%，而对照组的存活率为13%，RPS为4%。综上所述，我们的结果表明，含有杆状病毒表达的VP35-VP4蛋白的蚕蛹粉可诱导非特异性和特异性免疫反应，并保护草鱼免受GCRV感染，表明其可作为口服疫苗使用。
